As a large fraction of drug targets are membrane bound proteins (e.g., G-protein coupled receptors, ion-channels, etc.), there is a need for the development of surfaces that bind lipids incorporating membrane bound targets. For example, bilayer-lipid membranes adsorbed onto solid supports, referred to as supported bilayer-lipid membranes, can mimic the structural and functional role of biological membranes. See Bieri, C. et al., Nature Biotech, 1999, 17, 1105–1108; Groves, J. T. et al., Science 1997, 275, 651–653; Lang, H. et al., Langmuir 1994, 10, 197–210; Plant, A. L. et al., Langmuir 1999, 15, 5128–5135; and Raguse, B. et al., Langmuir 1998, 14, 648–659. These hybrid surfaces were developed to overcome the fragility of black lipid membranes while preserving aspects of lateral fluidity observed in native biological membranes. The properties of supported membranes are determined by the nature of the adsorbing surface. Self-assembled monolayers may be used to coat a substrate and are capable of further derivatization to attach membranes.
Surfaces binding lipid membranes can be broadly classified into three categories: (i) hydrophobic surfaces (e.g., self-assembled monolayers presenting terminal methyl groups) which support the adsorption of lipid monolayers are of limited utility as they cannot be used to incorporate membrane-spanning proteins (Plant, A. L., Langmuir 1999, 15, 5128–5135); (ii) hydrophilic surfaces (e.g., glass surfaces) which bind bilayer-lipid membranes are also of limited utility as they can only be used to incorporate membrane-spanning proteins with extra-membrane domains that are less thicker than the layer of adsorbed water (˜10° A) (Groves, J. T. et al., Science 1997, 275, 651–653; and Groves, J. T. et al., Langmuir 1998, 14, 3347–3350); and (iii) hybrid surfaces presenting amphiphilic anchor lipids that bind bilayer-lipid membranes offer the potential for incorporating a wide variety of membrane-spanning proteins (Lang, H. et al., Langmuir 1994, 10, 197–210; Raguse, B. et al., Langmuir 1998, 14, 648–659; and Vanderah, D. J. et al., Materials Research Society Fall Meeting Abstracts, Boston, 1999).
Self-assembled monolayers (“SAMs”) of alkanethiolates on gold are well suited for studying biomolecular recognition at surfaces because the well-defined structures are amenable to detailed characterization at a molecular level (e.g., Scanning Tunneling Microscopy “STM,” Atomic Force Microscopy “AFM,” etc.). See Widrig, C. A. et al., J. Am. Chem. Soc. 1991, 113, 2805–2810; and Alves, C. A. et al., J. Am. Chem. Soc. 1992, 114, 1222–1227. They may also be addressed by a variety of bioanalytical techniques (e.g., optical, electrochemical, etc.). See Lahiri, J. et al., Anal. Chem. 1999, 71, 777–790; Plant, A. L., Langmuir 1998, 14, 3347–3350; Rueda, M. et al., Langmuir 1999, 15, 3672–3678; and Steinem, C. et al., Bioelectrochem. and Bioenerg. 1997, 42, 213–220.
The importance of a hydrophilic spacer between the substrate and the adsorbed lipid has been studied. The use of thiolated anchor lipids consisting of dipalmitoylphosphatidic acid extended at the hydrocarbon end by a hydrophilic ethyleneoxy group linked at the other end to a terminal disulfide has been shown (Lang, H. et al., Langmuir 1994, 10, 197–210; Plant et al., Materials Research Society Fall Meeting Abstracts, Boston, 1999; and Raguse et al., Langmuir 1998, 14, 648–659). Similar anchor lipids containing thiaoligoethyleneoxide (HS(CH2CH2O)n−) moieties have also been used. However, these approaches have two disadvantages:
first, they require the laborious synthesis of the oligo(ethylene oxide) containing thiols, and second, the structures of the SAMs formed from these thiols may not be well-defined. Biotinylated anchor lipids have been used to immobilize streptavidin on self-assembled monolayers presenting biotin groups (Bieri, C. et al., Nature Biotech. 1999, 17, 1105–1108). Although this strategy circumvents issues regarding the structure and stability of putative self-assembled monolayers containing thiaoligoethyleneoxide moieties, the approach itself is cumbersome and requires the synthesis of biotinylated thiols. A simpler method for fabricating a supported membrane is desired.
Methods to create arrays of membranes would enable high-throughput screening of multiple targets against multiple drug-candidates. Arrays of membranes may be obtained by fabricating grids of titanium oxide on a glass substrate as titanium oxide resists the adsorption of lipids (Boxer, S. G. et al. Science 1997, 275, 651–653; and Boxer, S. G. et al. Langmuir 1998, 14, 3347–3350). Micropipeting techniques have been used to spatially address each corralled lipid-binding region (Cremer, P. S. et al., J. Am. Chem. Soc. 1999, 121, 8130–8131). However, these methods are cumbersome and require the fabrication of patterned surfaces. To make membrane arrays by printing membranes on unpatterned surfaces, it would be necessary to confine the membrane to the printed areas without lateral diffusion of the membrane molecules to the unprinted areas. Boxer et al. demonstrated that it was possible to pattern lipids on glass surfaces by microcontact printing using poly-dimethylsiloxane (PDMS) stamps “inked” with phosphatidylcholine (“PC”). They attributed the lateral confinement of the lipids to the stamped regions, to the self-limiting expansion of PC membranes to ˜106% of the original printed areas (Hovis, J. et al., Langmuir 2000, 16, 894–897).